JPH0689034B2 - CRF analog - Google Patents

Abstract

Rat Corticotropin Releasing Factor (rCRF) and the human counterpart thereof have the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu- Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met- Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His- Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. Analogs are disclosed that are at least as potent. One which has been found to be particularly potent is: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr- Phe-His-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met- Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala- Ala-Leu-Asn-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH2. These analogs or pharmaceutically or veterinarily acceptable salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier, can be administered to mammals, including humans, to achieve a substantial elevation of ACTH, beta -endorphin, beta -lipotropin, other products of the pro-opiomelanocortin gene and corticosterone levels and/or a lowering of blood pressure over an extended period of time. They may also be used as stimulants to elevate mood and improve memory and learning, as well as diagnostically. <??>In the analogs, one or more of the first three N-terminal residues may be deleted or may be substituted by a peptide up to 10 amino acids long and/or by an acylating agent containing up to 7 carbon atoms. A number of other substitutions may also be made throughout the chain.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to rat CRF (rCRF) and pharmaceutical compositions comprising rCRF.

The concept that the hypothalamus plays an important role in the secretory function of adrenocortical stimulator cells in the anterior pituitary has been supported by experimental and clinical observations. Over 25 years ago, Gu
illemin, Rosenberg, Saffran and Schally each independently determined that there was a factor in the hypothalamus that increased the amount of ACTH secreted from the pituitary that was incubated in vitro or the pituitary that was maintained in organ culture. Sheep CRF (oCRF) is 198 for all characterized secretagogues.
Until characterized one year, it did not meet the expected criteria for physiological corticotropin-releasing factor (CRF). The oCRF has the following formula as disclosed in U.S. Pat. No. 4,415,558: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lvs-Ala-As
p-Gln-Leu-Ala-Glu-Glu-Ala-His-Ser-Asn-Arg-Lys-Leu-
It was found to be represented by Leu-Asp-Ile-Ala-NH ₂ .

Sauvagine is a nearly similar amidated peptide of 40 amino acid residues isolated from the skin of the South American frog Phyllomedus asauvagei. This is characterized by Erspamer et al., Regulatory Peptides, Volume 2, 1-
It is described on page 13 (1981). Saubadin has the following formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser- Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gl.
n-Glu-Lys-Glu-Lys-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-
It is represented by Leu-Asp-Thr-Ile-NH ₂ . Saubadin and oCRF were reported to have the biological activities of lowering blood pressure in mammals and stimulating the secretion of ACTH and β-endorphin.

SUMMARY OF THE INVENTION Rat CRF (rCRF) is now isolated, purified, and of the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
It was characterized to be a Hentetracontapeptide represented by Met-Glu-Ile-Ila-NH ₂ . This is also known as rat Amunine
Is called. The structural formula of human CRF was found to be the same as that of rCRF. Synthesis of the 41-amino acid residue peptide is complete, and both isolated and synthetic rCRF stimulate ACTH and β-endorphin activity in vitro and in vivo. It has been found that synthetic rCRF substantially lowers blood pressure over an extended period of time. As a result, the synthetic rC
RF is available in essentially pure form (ie, essentially free of crude biological extract and remnants and related synthetic replicates), and is a synthetic compound with a purity of at least about 5%. (Which is essentially more pure than the naturally occurring peptide) is considered to have utility. In fact, it is possible to obtain at least about 90% or higher purity, which will be used for clinical trials.

An analogue of a 41-amino acid residue CRF peptide of the formula or their non-toxic addition salts have at least substantially the same biological activity: YR ₁ -Pro-Pro-Ile-Ser-R ₈ -R _9- leu-R _11- R _12- R _13- l
eu-leu-Arg-R ₁₇ ‐R ₁₈ ‐R ₁₉ ‐Glu-R ₂₁ ‐R ₂₂ ‐R ₂₃ ‐R ₂₄ ‐
_{R 25 -R 26 -R 27 -R 28} -R 29 -Gln-ala-R 32 -R 33 -Asn-Arg
-R ₃₆ -R ₃₇ -R ₃₈ -R ₃₉ -R ₄₀ -R ₄₁ -NH _{2 In the} formula, Y is an acyl group having 7 or less carbon atoms or a hydrogen atom; R ₁ is Ser-Gln-Glu, pGlu -Gly, Gln-Glu, Glu, D-Ser-Gln-Gl
u, Ser-Glu-Glu, D-Ser-Glu-Glu, Glu-Glu, D-pGlu-Gly or des R ₁ ; R ₈ , R ₁₂ , R ₁₉ and R ₂₄ are leu, Ile, ala, Gly, Val, Nle, Phe
R ₉ is Asp or Glu; R ₁₁ is Thr or Ser; R ₁₃ is His, Tyr or Glu; R ₁₇ is Glu or Lys; R ₁₈ is Val, Nle or Met; R ₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R ₂₂ is ala, Thr, Asp or Glu; R ₂₃ is Arg, Orn , Har or Lys; R ₂₅ is Asp or Glu; R ₂₆ is Gln, Asn or Lys; R ₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R ₂₈ is ala, Arg or Lys; R ₂₉ is Gln or Glu; R ₃₂ is His, Gly, Tyr or ala; R ₃₃ is Ser, Asn, len, Thr or ala; R ₃₆ is Lys, Orn, Arg, Har or Leu; R ₃₇ is leu or Tyr; R ₃₈ is Met or leu; R ₃₉ is Glu or Asp; R ₄₀ is Ile , Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or
Gln; R ₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or Death R ₄₁
Provided that R ₂₂ is Ala and / or R ₃₃ is leu when R ₃₈ is Leu.

The pharmaceutical composition according to the invention comprises rCRF or a non-toxic addition salt thereof dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier. Administration of the peptides according to the invention or their pharmaceutically or veterinarily acceptable addition salts to mammals (especially humans) is
-To regulate the secretion of endorphin, β-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone,
And / or to lower blood pressure and / or to affect mood, behavior and gastrointestinal function and activity of the autonomic nervous system. In addition, CRF analogs can be used to assess the functional status of the pituitary gland, cardiovascular system, gastrointestinal system or central nervous system.

DISCLOSURE OF THE INVENTION The nomenclature used to define peptides is Schroder.
And Lubke, "The Peptides," published by the Academic Press (1965), where by convention the amino group is on the left and the carboxyl group is on the right. Standard three letter abbreviations are used to indicate α-amino acid residues, and where an amino acid residue has isomers, it is the L form of the indicated amino acid unless otherwise indicated: Ser = L-serine,
Nie = L-norleucine, Nva = norvaline, Har = homoarginine, Orn = ornithine and the like. The following abbreviations are also used: either leu = L-leucine or C CH ₃ -L-leucine (CML), and ala = L-alanine or C CH ₃ -L-alanine (CMA). On the other hand.

CRF analogs are represented by the formula (I): YR ₁ -Pro-Pro-Ile-Ser-R ₈ -R ₉ -leu-R ₁₁ -R ₁₂ -R ₁₃ -l
eu-leu-Arg-R ₁₇ ‐R ₁₈ ‐R ₁₉ ‐Glu-R ₂₁ ‐R ₂₂ ‐R ₂₃ ‐R ₂₄ ‐
_{R 25 -R 26 -R 27 -R 28} -R 29 -Gln-ala-R 32 -R 33 -Asn-Arg
-R ₃₆ -R ₃₇ -R ₃₈ -R ₃₉ -R ₄₀ -R ₄₁ -NH _{2 In the} formula, Y is an acyl group having 7 or less carbon atoms or a hydrogen atom; R ₁ is Ser-Gln-Glu, pGlu -Gly, Gln-Glu, Glu, D-Ser-Gln-Gl
u, Ser-Glu-Glu, D-Ser-Glu, Glu, Glu-Glu, D-pGlu-Gly or des R ₁ ; R ₈ , R ₁₂ , R ₁₉ and R ₂₄ are leu, Ile, ala, Gly, Val, Nle, Phe
R ₉ is Asp or Glu; R ₁₁ is Thr or Ser; R ₁₃ is His, Tyr or Glu; R ₁₇ is Glu or Lys; R ₁₈ is Val, Nle or Met; R ₂₁ is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R ₂₂ is ala, Thr, Asp or Glu; R ₂₃ is Arg, Orn , Har or Lys; R ₂₅ is Asp or Glu; R ₂₆ is Gln, Asn or Lys; R ₂₇ is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R ₂₈ is ala, Arg or Lys; R ₂₉ is Gln or Glu; R ₃₂ is His, Gly, Tyr or ala; R ₃₃ is Ser, Asn, len, Thr or ala; R ₃₆ is Lys, Orn, Arg, Har or Leu; R ₃₇ is leu or Tyr; R ₃₈ is Met or leu; R ₃₉ is Glu or Asp; R ₄₀ is Ile , Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or
Gln; R ₄₁ is ala, Ile, Gly, Val, Leu, Nle, Phe, Gln or Death R ₄₁
Where R ₂₂ is Ala and / or R ₃₈ is Leu
R ₃₃ is leu.

These synthesized analogs are at least as potent as native CRF. The present invention has the following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
Provides rCRF represented by Met-Glu-Ile-Ile-NH ₂ .

Peptides are synthesized by any suitable method, such as exclusive solid phase, partial solid phase, fragment condensation or classical solution addition. Certain CRF analogs that do not contain D-isomer residues or non-naturally occurring amino acid residues can be synthesized by the recently developed recombinant DNA method.

It should be understood that synthesis by use of recombinant DNA methods encompasses, for the purposes of this application, the appropriate use of structural genes which encode the intended form of the CRF analog. Synthetic CRF peptides are transformed into microorganisms using expression vectors containing promoters and operators along with such structural genes, and transformed into the transformed microorganisms.
Obtained by expressing the CRF peptide. Also,
Non-human animals also have such structural genes and US patents.
Genetic engineering using the general method described in 4276282 (issued June 30, 1981), or WO83 / 01783 (May 1983)
Issued on December 26th) and WO82 / 04443 (issued December 23rd, 1982)
CRF peptides can be used to produce CRF peptides by genetic engineering using microinjection of early embryos as described in. The synthetic CRF peptide is then appropriately recovered from the animal, such as by extraction from serum.

Protecting the labile side chains of various amino acid moieties with suitable protecting groups, which serve to prevent chemical reactions from occurring at the site until the protecting group is finally removed, is the It is usual in chemical synthesis. In addition, the α-amino group of amino acids or peptide fragments is protected, during which the substance reacts at the carboxyl group, and then the α-amino protecting group is selectively removed to allow the next reaction at that site. It is also common to have them do so. Therefore, as one step of peptide synthesis, it is not possible to produce an intermediate compound consisting of each amino acid residue arranged at an intended sequence position in the peptide chain using various amino acid residues having a side chain protecting group. That is normal.

The intermediate is represented by the following formula (II): X ^1- R ₁ -Pro-Pro-Ile-Ser (X ² ) -R ₈ -R ₉ (X ⁵ ) -leu-R
₁₁ (X ² ) -R ₁₂ (X ⁴ ) -R ₁₃ (X or X ⁵ ) -leu-leu-Ar
_{^{g (X 3) -R 17 (}} X 5 or _{^{X 6) -R 18 - 19 (}} X 4) -Glu
_{^{(X 5) -R 21 -R 22}} (X 2 or X ⁵⁾ -R ₂₃ (X ³ or X ⁶⁾
‐R ₂₄ (X ⁴ ) ‐R ₂₅ (X ⁵ ) ‐R ₂₆ (X ⁴ or X ⁶ ) ‐R ₂₇ (X ⁴
Or X ⁵ ) -R ₂₈ (X ³ or X ⁶ ) -R ₂₉ (X ⁴ or X ⁵ )-
Gln (X ⁴ ) -Ala-R ₃₂ (X) -R ₃₃ (X ² or X ⁴ ) -Asn
^{(X 4) -Arg (X 3} ) -R 36 (X 6) -R 37 (X) -R 38 -R 39
(X ⁵ ) -R ₄₀ (X ² or X ⁴ or X ⁵ ) -R ₄₁ (X ⁴ ) -X ^{7 In the} formula, the R group is as defined above.

X ¹ is a hydrogen atom or an α-amino protecting group. The α-amino protecting groups contemplated by X ¹ are known to be useful in the art of step-wise synthesis of polypeptides.
The α-amino protecting group included in X ¹ includes (1) an acyl-type protecting group such as a formyl group and an acrylyl group (Ac
r), benzoyl groups (Bz) and acetyl groups (Ac), these protecting groups are preferably used only at the N-terminus.
(2) Aromatic urethane type protecting groups such as benzyloxycarbonyl group (Z) and substituted Z (eg p-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group and p-methoxy) Benzyloxycarbonyl group);
(3) Aliphatic urethane type protecting group such as t-butyloxycarbonyl group (BOC), diisopropylmethoxycarbonyl group, isopropyloxycarbonyl group, ethoxycarbonyl group and allyloxycarbonyl group; (4)
Cycloalkyl urethane type protecting groups such as fluorenylmethyloxycarbonyl group (FMOC), cyclopentyloxycarbonyl group, adamantyloxycarbonyl group and cyclohexyloxycarbonyl group; and (5)
A thiourethane type protecting group such as a phenylthiocarbonyl group; The preferred α-amino protecting group is BOC.

X ² is a protecting group for the hydroxyl group of Thr and Ser, and is acetyl group (Ac), benzoyl group (Bz), t-butyl group, triphenylmethyl group (trityl group), tetrahydropyranyl group, benzyl ether. Group (Bzl) and 2,6
Advantageously selected from the group consisting of the dichlorobenzyl group (DCB). The optimal protecting group is Bzl. X ² can also be a hydrogen atom, in which case there is no protecting group on the hydroxyl group.

X ³ is a protecting group for the guanidino group of Arg or Har, and is a nitro group, p-toluenesulfonyl group (Tos),
It is advantageously selected from the group consisting of Z, an adamantyloxycarbonyl group and BOC or is a hydrogen atom. Tos is the best.

X ⁴ is a hydrogen atom or a protecting group for the amide group of Asn and Gln, preferably a xanthyl group (Xan).

X ⁵ is a hydrogen atom or an ester-forming protecting group for the β- or α-carboxyl group of Asp and Glu, and is derived from benzyl ester, 2,6-dichlorobenzyl ester, methyl ester, ethyl ester and t-butyl ester. Advantageously selected from the group OBzl is the best choice.

X ⁶ is a hydrogen atom or a protecting group for the side chain amino group of Lys and Orn. Examples of suitable side chain amino protecting groups are Z, 2
-Chlorobenzyloxycarbonyl group (2-Cl-Z),
Tos, t-amyloxycarbonyl group (Aoc), BOC and the aromatic or aliphatic urethane type protecting groups specified above.

When His is present, X is a hydrogen atom or a protecting group for the imidazole nitrogen, such as Tos and 2,4-dinitrophenyl group (DNP), and when Tyr is present, X
Is a protecting group for a hydrogen atom or a hydroxyl group, for example DCB. If Met is present, its sulfur atom may optionally be protected with an oxygen atom.

The choice of side chain amino protecting group is not critical, provided that the protecting group is not removed during the synthesis by the leaving reaction of the α-amino protecting group. Therefore, the α-amino protecting group and the side chain amino protecting group must not be the same.

X ⁷ is NH ₂ , a protecting group such as an ester or anchorage bond used in solid phase synthesis to attach to a solid resin support,
Preferably the following formula: -NH-benzhydrylamino (BHA)
A resin support and a -NH-paramethylbenzhydrylamine (MBHA) resin support. Cleavage from the BHA or MBHA resin directly gives the CRF analog amide. By using a methyl derivative of such a resin, a methyl-substituted amide can be produced.

In the formulas for the above intermediates X, X ¹ , X ² , X ³ , X ⁴ , X ⁵
And at least one of X ⁶ is a protecting group. Each R
The particular amino acid selected for the group is determined to attach the protecting groups generally known in the art identified above. The following rules are followed in selecting individual side chain protecting groups for use in peptide synthesis: (a) the protecting group is a reagent selected to remove the α-amino protecting group at each step of the synthesis and Should be stable under the reaction conditions,
(B) The protecting group should retain its protective properties and should not be cleaved under coupling conditions, and (c) the side chain protecting group should not denature the peptide chain at the completion of the synthesis involving the intended amino acid sequence. It should be removable under the reaction conditions.

For the N-terminal acyl group represented by Y, an acetyl group, a formyl group, an acrylyl group and a benzoyl group are preferable. Any amino acid can be used for a 1 to 10 amino acid peptide (this peptide is optionally included without adversely affecting potency), but normally the naturally occurring L or D form of the amino acid is used. To be

Thus, it is envisaged that the present invention also provides a process for the preparation of compounds of formula (I) which comprises (a) a peptide having at least one protecting group and of formula (II) Medium, X, X ¹ , X ² , X ³ , X ⁴ , X ⁵ and
X ⁶ is each a hydrogen atom or a protecting group, and X ⁷ is a protecting group, an anchor bond of the resin support, OH or NH ₂ ); (b) one from the above peptides of formula (II) Or cleaving further protecting groups or anchoring bonds; and (c) optionally converting the resulting peptide to its non-toxic addition salt.

When peptides are produced by chemical synthesis, they are
It can be conveniently prepared using solid-phase synthesis methods such as those described by J. Am. Chem. Soc., 86, 2149 (1964) by ield, but other similar chemistries known in the art. Synthetic methods are also used as previously described. Solid phase synthesis is described in US Pat. No. 4,244,946 by Rivier et al. (Issued Jan. 21, 1981,
The disclosure is initiated at the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin, as is generally described in (herein incorporated by reference). Such starting material for rCRF is prepared by attaching an α-amino-protecting group Ile to a BHA resin.

BOC protected Ile is coupled to BHA resin using methylene chloride and dimethylformamide (DMF). After coupling BOC-Ile to the resin support, α-
The amino protecting group is trifluoroacetic acid (TF
A), TFA alone or by using HCl in dioxane. Advantageously 50% by volume in methylene chloride
TFA is used with 0-5% by weight of 1,2-ethanedithiol. This protecting group removal reaction is carried out at about 0 ° C to room temperature. Other standard cleavage reagents for the removal of specific α-amino protecting groups as well as their reaction conditions are described in Schroder.
And "The Peptides" by Lubke, pages 1,72-75 (Academic Press, 1965).

After removal of the α-amino protecting group of Ile, the remaining α-amino- and side chain-protected amino acids are stepwise coupled in the intended order to give the intermediate compound as defined above.
Instead of adding each amino acid separately to the synthesis reaction, some of those amino acids may be coupled to each other before addition to the solid phase reactor. Selection of the appropriate coupling agent is known to those skilled in the art. Particularly suitable coupling agents are N, N'-dicyclohexylcarbodiimide (DCCI) and N, N'-diisopropylcarbodiimide (DICI).

Activating reagents used in solid phase synthesis of peptides are well known in the peptide art. Examples of suitable activation reagents include
There are carbodiimides such as N, N'-diisopropylcarbodiimide and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide. For other activating reagents and their use in peptide coupling, see Chapter III of the same book by Schroder and Lubke and J. Phar . Sci ., 59, 1-27 (1970) by Kapoor.
It is described in.

Each protected amino acid or amino acid sequence was introduced into the solid phase reactor in a 4-fold excess, and the coupling was carried out in a mixed medium of dimethylformamide (DMF): CH ₂ Cl ₂ (1: 1) or DM.
It is carried out in a single medium of F or CH ₂ Cl ₂ . When the coupling is done manually, the completion of the coupling reaction at each step of the synthesis is described by E. Kaiser et al. Anal. Bioche.
m ., 34,595 (1970) and monitored by the ninhydrin reaction. If incomplete coupling occurs, α before coupling the next amino acid
The coupling method is repeated before removal of the amino protecting group. The coupling reaction is, for example, Beckma.
n) 990 automatic synthesizer, Biopolymers , 197 by Rivier et al.
It can be done automatically using a program such as that reported on pages 8,17,1927 to 1938.

After completion of the intended amino acid sequence, the intermediate peptide may be treated with a reagent such as liquid hydrogen fluoride (which not only cleaves the peptide from the resin, but also leaves the remaining side chain protecting groups X ² , X ³ ,
X ⁴ , X ⁵ and X ⁶ and / or X ¹ is a reagent that also cleaves off its α-amino protecting group X ¹ if it is not the acyl group intended to be present in the final peptide). Cleaved from the support to yield the peptide. When hydrogen fluoride is used to cleave off, anisole or cresol and methyl ethyl sulfide are included in the reaction vessel as scavengers. If the Met is present in the sequence, its BOC protecting group can be cleaved with trifluoroacetic acid (TFA) / ethanedithiol to eliminate S-alkylation prior to cleavage of the peptide from the resin.

The following example illustrates a preferred method for synthesizing CRF analogs by the solid phase method.

Example I The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
The synthesis of rCRF represented by Met-Glu-Ile-Ile-NH ₂ was performed by Bachem with substitution range of about 0.1-0.5 mmol / g of resin.
Performed by the stepwise method on MBHA hydrochloride resin commercially available from the company. The synthesis was carried out on a Beckman 990B Peptide Synthesizer using a suitable program, preferably the following program.

Coupling of BOC-Ile resulted in about 0.35 mmol of Ile displacement per gram of resin. All solvents used were degassed by bubbling with an inert gas such as helium or nitrogen to eliminate unwanted oxygen as it oxidizes the sulfur atoms of the Met residue.

After removal of the protecting groups and neutralization, the peptide chains were coupled onto the resin step by step. Generally, 1 to 2 mmol of BOC-in methylene chloride per gram of resin in 2 hours.
Protected amino acids and 1 equivalent of 2 molar DCCI in methylene chloride were used. A 50% mixture of DMF and methylene chloride was used when coupling BOC-Arg (Tos). Bzl
Was used as a hydroxyl side chain protecting group for Ser and Thr. p-nitrophenyl ester (ONp) is used to activate the carboxyl end of Asn or Gln,
And for example BOC-Asn (ONp) was coupled overnight using 1 equivalent of HOBt in a 50% mixture of DMF and methylene chloride. The amide group of Asn or Gln was protected with Xan when DCCI coupling was used instead of the active ester method. 2-Cl-Z was used as a protecting group for the Lys side chain. Tos was used to protect the guanidino group of Arg and the imidazole group of His, and the side chain carboxyl group of Gln or Asp was protected with OBzl. At the end of synthesis, the following formula: BOC-Ser (Bzl) -Glu (OBzl) -Glu (OBzl) -Pro-Pro-
Ile-Ser (Bzl) -Leu-Asp (OBzl) -Leu-Thr- (Bzl)-
Phe-His (Tos) -Leu-Leu-Asp (Tos) -Glu (OBzl) -V
al-Leu-Glu (OBzl) -Met-Ala-Arg (Tos) -Ala-Glu (O
Bzl) -Gln (Xan) -Leu-Ala-Gln (Xan) -Gln (Xan)
-Ala-His (Tos) -Ser (Bzl) -Asn (Xan) -Arg (To
s) -Lys (2-Cl-Z) -Leu-Met-Glu (OBzl) -Ile-Ile-
An intermediate compound represented by the resin support was obtained. Xan could be partially or completely removed by the TFA treatment used to remove the α-amino protecting group.

In order to separate the resulting protected peptide-resin and remove the protecting group, 1.5 m of anisole per 1 g of peptide-resin was used.
l, methyl ethyl sulfide 0.5 ml and hydrogen fluoride (HF)
Using 15 ml, first at −20 ° C. for 20 minutes, then at 0 ° C. for 30 minutes
Processed for a minute. After exclusion of HF under high vacuum, the peptide-resin was alternately washed with dry diethyl ether and chloroform, after which the peptide was extracted with degassed 2N acetic acid in water and separated from the resin in time.

Peptides were purified by gel permeation chromatography and then Peptides: Structure and Biological F by Rivier et al.
For semi-preparation as described in unction , pages 125-1238 (1979) and J. Chromatography (1983) by Rivier et al.
Purified by HPLC. The chromatographic fractions were carefully monitored by HPLC and only the essentially pure fractions were collected.

The specific optical rotation of the rCRF peptide synthesized and purified by the above method was measured by Perkin Elmer type 141 ▲ [α] ²² _D ▼ = 51.4 ± 1.0 (c = 1 in 1% acetic acid)
(Corrected for the presence of H ₂ O and TFA: −93.5 ° ±
1.0) and its purity was about 95%.

To determine if the exact amino acid sequence intended was achieved, the rCRF peptide was placed in a sealed exhaust tube containing boiling HCl, 3 μl / ml thioglycol and 1 nmol Nle (as internal standard) at 140 ° C. for 9 hours. It was hydrolyzed. Amino acid analysis of the hydrolyzate (Beckman 12
1MB amino acid analyzer used) has the following amino acid ratio: Asx
(1.9), Thr (0.8), Ser (3.1), Glx (9.0), Pro (2.
1), Ala (3.8), Val (0.9), Net (1.9), Ile (2.6), Le
u (7.0), Phe (0.9), Lys (1.0), His (2.O) and Arg
(3.0) was confirmed, which confirmed that a 41-amino acid residue peptide was obtained.

Example II rCRF was extracted, isolated and purified by the following method. Lyophilized rat hypothalamus was defatted with acetone and the resulting powder was treated with 10 volumes of a mixture containing 1N acetic acid (HOAc), 0.1H HCl, 0.5% β-mercaptoethanol, 10 mmol EDTA and 5 μg / ml pepstatin A. It was extracted at a temperature of 90 ° C or higher. The warm slurry was immediately milled in a blender, cooled in an ice bath, and centrifuged. The supernatant was set aside and the precipitate was extracted again with the above mixture with the addition of 20 mM NaCl. The combined supernatants were defatted by multiple extractions with 2 volumes of a mixed solvent of ethyl ether / petroleum ether (1: 2).

The aqueous phase is a Pharmacia K215 / 100 column (85c
m Sephadex G-50 was subjected to gel perchromatography at 4 ° C. on a column packed with 5 cm Sephadex G-10, V _T = 311). Eluent is 0.2% β
-3N HOAc containing mercaptoethanol. Corticotropin / β-endorphin-releasing factor bio- and immuno-activity co-eluted with GH-releasing activity in the Kav = 0.20-0.31 region. The CRF band is J. Liquid Ch by Rivier
Waters Assoc with a Vydac C ₁₈ filled cartridge as described in romat.1: 343-367,1978.
Further purification by preparative HPLC using the iates Prep 500 system and triethylammonium phosphate (TEAP) / acetonitrile buffer system. The active fraction was further purified by HPLC using a Vydac C ₄ semipreparative column and a trifluoroacetic acid / acetonitrile system.

Then, rCRF was purified. Analytical separations were first performed on a Vydac diphenyl (5μ) column using a TEAP / CH ₃ CN gradient. Next, 0.1 with a Vydac C ₁₈ (5μ) column.
% Using TFA / CH ₃ CN gradient was continuously analytical separation twice. Finally, with a Vydac diphenyl (5μ) column,
It was added separating two times using 1% TFA / CH ₃ CN gradient.
About 2 nmol rCRF (purity about 90%) was obtained in the last step. Composition as well as structural analysis gave the following sequences.

H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Glu-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
Met-Glu-Ile-Ile-NH ₂ Example III For synthetic and natural rCRF, their in vitro
The effect on the secretion of ACTH and β-endorphin was tested. The synthetic rCRF was also tested in vivo. ACTH and β- by cultured rat pituitary cells
The high potency of synthetic and natural rCRF that stimulates endorphin secretion was measured using the method generally described in Endocrinology , 91, 562 (1972). The minimum and half-maximal responses are about 10 pmol each of synthetic rC.
RF and about 100 pmol of synthetic rCRF were observed. The secretory response to the highest concentration of rCRF (5 nmol) was plateau-level. Science vivo tests by C.Bivier like, 21
Performed using the general method described on page 8,377 (1982). The dose of 30 ng to 3 μg per 1 kg of body weight is a generic term for ACTH and β-endorphin-like substances (substances showing immunoreactivity similar to β-endorphin,
-Including endorphins and acylated β-endorphins. Hereinafter, the secretion of β-END-LI) is rapidly increased to 5
~ 20 times higher.

Synthetic rCRF is a potent ACTH and β-END in vivo in rats
-It was found to be a LI secretion stimulant. Plasma levels of ACTH and β-END-LI were at least 5-20 minutes after intravenous cannulation of Nembutal anesthetized male and quiescent male or female rats via intravenous rCRF. Rose. Moreover, rCRF was found to have a dramatic effect in lowering blood pressure in rats and dogs.

The acute toxicity test of rCRF was performed as follows.

Male Balb-C mice weighing 20 to 25 g were divided into the following 4 groups, and 0.9
Vehicle containing 1% 10 ⁻⁴ ascorbic acid in saline or 1 mg / ml of CRF was administered.

A 8 animals V ¹⁾ Subcutaneous anesthesia B 8 animals CRF ²⁾ Subcutaneous anesthesia C 8 animals V Intravenous ether anesthesia D 8 animals CRF Intravenous ether anesthesia 1) V: Vehicle 2) CRF: 10 mg CRF / kg body weight Group A and None of the mice in Group B showed any abnormality, and all survived after 18 hours.

The mice in groups C and D rapidly awakened from light ether anesthesia and exhibited normal post-anesthesia behavior. All the mice survived after 18 hours.

From the above results, it was shown that CRF has no acute toxicity to mice at the dose of 10 mg / kg body weight.

When 0.01 to 5 μg / kg body weight of CRF was administered to healthy humans, no serious side effects occurred at any concentration.

Reference Example I The following formula: Ac-Gly-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe
-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-G
lu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu
A peptide [acetyl-Gly ¹ ] -rCRF represented by -Met-Glu-Ile-Ile-NH ₂ was synthesized. Testing by the general method shown in Example III showed that this peptide also stimulated secretion of ACTH and β-END-L1.
And it was revealed that blood pressure was lowered significantly.

Reference Example II The following formula: H-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-
Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Al
a-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-
Peptide represented by Ile-NH ₂ [Des Ser ^1- Glu ^2- Glu ³ ] -rCRF
Was synthesized. Studies by the general method described in Example III revealed that this peptide also stimulates the secretion of ACTH and β-END-LI and significantly reduces blood pressure.

Reference Example III The following formula: H-Tyr-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-
Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Al
a-Glu-Glu-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-
A peptide [Tyr-Ser ¹ ] -rCRF represented by Leu-Met-Glu-Ile-Ile-NH ₂ was synthesized.
Studies by the general method described in Example III revealed that this peptide also stimulates the secretion of ACTH and β-END-LI and significantly reduces blood pressure.

Reference Example IV The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Ala-Glu-Met-Thr-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
A peptide represented by Met-Glu-Ile-Ile-NH ₂ [Ala ¹⁹ . Thr ²² ] -rCRF was synthesized. Testing by the general method described in Example III showed that this peptide also stimulated secretion of ACTH and β-END-LI,
And it was proved that blood pressure was lowered significantly.

Reference Example V Formula: Acr-Leu-Gly-Val-Ser-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Le
u-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-
Arg-Ala-Glu-Glu-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Ar
Peptide represented by g-Lys-Lea-Met-Glu-Ile-Ile-NH ₂ [acrylyl-Leu-Gly-Val ¹ , Se
r ² ] -rCRF was synthesized. Testing by the general method described in Example III showed that this peptide also showed ACTH and β-END-L.
It was demonstrated to stimulate the secretion of I and reduce blood pressure fairly significantly.

Reference Example VI The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
Glu-Leu-Leu-Arg-Glu-Val-Leu-Glu-Val-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
The peptide [Glu ¹³ , Val ²¹ ] -rCRF represented by Met-Glu-Ile-Ile-NH ₂ was synthesized. Testing by the general method described in Example III showed that this peptide was significantly more active than rCRF itself and β to ACTH.
It was demonstrated to stimulate the secretion of -END-LI and reduce blood pressure fairly significantly.

Reference Example VII The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Nle-Asp-Leu-Ser-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-
A peptide [Nle ⁸ , Ser ¹¹ , Leu ³³ ] -rCRF represented by Met-Glu-Ile-Ile-NH ₂ was synthesized. This peptide was also demonstrated by the general method described in Example III to stimulate ACTH and β-END-LI secretion and to significantly significantly reduce blood pressure.

Reference Example VIII Formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Ala-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
Peptide represented by Leu-Glu-Ile-Nle-NH ₂ [Ala ²¹ , Leu ³⁸ , Nle ⁴¹ ] -rCRF
Was synthesized. This peptide was also demonstrated by the general method described in Example III to stimulate ACTH and β-END-LI secretion and to significantly significantly reduce blood pressure.

Reference Example IX The following formula: Bz-Gly-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Nle-His
-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-G
ln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met
-Glu-Ile-Ile-NH ₂ peptide [benzoyl-Gly ¹ , des Gln ³ ,
Nle ¹² ] -rCRF was synthesized. This peptide was also demonstrated by the general method described in Example III to stimulate ACTH and β-END-LI secretion and to significantly significantly reduce blood pressure.

Reference Example X The following formula: Ac-D-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-P
he-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala
-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-L
Peptide represented by eu-Met-Asp-Ile-Ile-NH ₂ [acetyl-D-Ser ¹ , Asp ³⁹ ] -r
CRF was synthesized. This peptide was prepared according to the general method described in Example III to a greater extent than rCRF itself.
It was demonstrated to stimulate the secretion of END-LI and to reduce blood pressure fairly significantly.

Reference Example XI The following formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Lys-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
Leu-Glu-Ile-Ile-NH₂ The peptide represented by [Gln²， Lys^{twenty three}， Leu 38] -rCRF
Synthesized. This peptide was prepared according to the general method described in Example III.
Similarly stimulates the secretion of ACTH and β-END-LI.
, And proved to lower blood pressure fairly significantly
It was

Reference Example XII Formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Gl
u-Glu-Leu-Ala-Glu-Glu-Ala-Tyr-Ser-Asn-Arg-Lys-Leu-
A peptide [Nle ²¹ , Tyr ³² ] -rCRF represented by Met-Glu-Ile-Ile-NH ₂ was synthesized. This peptide was labeled by the general method described in Example III.
It was demonstrated to stimulate the secretion of ACTH and β-END-LI to a greater extent than CRF itself, and to significantly significantly lower blood pressure.

Reference Example XIII The following formula: H-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-
Arg-Lys-Met-Ile-Glu-Ile-Ala-Lys-Gln-Glu-Lys-Glu-Ly
s-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Met-Asp-Thr-
Peptide represented by Ile-NH ₂ [des pGlu ^1- Gly ² , Ala ²¹ , Met
³⁷ ] -Sauvazine was synthesized. This peptide is described in Example II.
Same as ACTH and β-END-LI according to the general test method described in I.
Has been demonstrated to stimulate the secretion of erythrocytes, and to significantly reduce blood pressure.

Reference Example XIV The following formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-L
eu-Leu-Arg-Lys-Met-Ile-Glu-Ala-Arg-Lys-Gln-Glu-Lys
-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Asp-I
le-Ile-NH ₂ peptide represented by ^{[Ala 21, Arg 22, Ile} 39,40] - Saubajin was synthesized. This peptide was also demonstrated by the general test method described in Example III to stimulate the secretion of ACTH and β-END-LI and to significantly significantly lower blood pressure.

Reference example XV Formula: pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-L
eu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Gln-Lys
-Leu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Met-A
A peptide [Leu ²⁶ , Met ³⁷ ] -sauvadin represented by sp-Thr-Ile-NH ₂ was synthesized. This peptide was also demonstrated by the general method described in Example III to stimulate ACTH and β-END-LI secretion and to significantly significantly reduce blood pressure.

Since CRF analogs exhibit marked hypotension, they will be of particular value in the treatment of hypertensive conditions and in the treatment of patients undergoing certain types of surgery.

CRF strongly stimulates the pituitary-adrenocortical axis male and therefore CRF
The analogs may be useful in stimulating the function of this axis in certain patients with poor glucocorticoid production in the body. For example, CRF restores pituitary-adrenocortical function to patients with pituitary-adrenocortical function remaining depressed and undergoing in vitro glucocorticoid treatment. Would be useful to.

Most other regulatory peptides have been found to affect the central nervous system and the gastrointestinal tract. ACTH and β-EN
Since D secretion is a "requisite" of the mammalian response to stress, it was expected that CRF would have a significant effect on the brain as a mediator of the body's stress response. Therefore, CRF is the mood of a person with normal but disturbed mind,
It may be used in learning or improving behavior. CRF
Since the analogues increase the secretion of ACTH, β-END, β-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone, its administration induces their action in the brain and its surroundings, which Can affect memory, mood, pain perception, and more specifically, excitement, depression and / or anxiety. For example, when administered to the ventricles, CRF can act as a natural stimulant by enhancing rat activity and improving learning behaviour.

CRF analogs may also be used to increase blood flow to the gastrointestinal tract of mammals (especially humans). All CR
F-related peptides were found to penetrate the mesenteric vascular bed. In addition, oCRF suppresses gastric acid production, so CR
F analogs are expected to be effective in treating gastric tumors in mammals by reducing gastric acid production and / or reducing gastrointestinal function.

CR in combination with a pharmaceutically or veterinary acceptable carrier
The F analog or its non-toxic bioaddition salt can be administered to mammals including humans intravenously, subcutaneously, intramuscularly, transdermally (for example, intranasally),
It is given in the cerebrospinal or orally. The peptide should be at least about 90% pure, preferably at least about 98% pure. However, those having a lower purity are also effective and can be used in mammals other than humans. This purity means that the intended peptide constitutes the above weight% of all similar peptides and peptide fragments present. Administration by humans for the purpose of lowering blood pressure or stimulating glucocorticoid production in the body is performed by a doctor. The required dose will vary with the particular condition being treated, the degree of the condition and the duration of the treatment.

These peptides can also be used to test the functions of the hypothalamus, pituitary gland, and adrenal gland in mammals with suspected endocrine or central nervous system disorders by monitoring physical function after administration of the peptides. used. For example,
It is administered as a diagnostic agent to test for Cushing's disease and mental disorder (eg, depression).

Such peptides may be pharmaceutically or veterinarily acceptable non-toxic salts such as acid addition salts or metal complexes with zinc, iron, calcium, barium, magnesium, aluminum, etc. Regarded as)
Often administered in the form of. Examples of acid addition salts include hydrochloride,
Hydrobromide, sulfate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, apple Acid salts, ascorbates and tartrates. When the active ingredient is administered in the form of tablets, the tablets may contain binders (eg trigger cant, corn starch or gelatin); disintegrants (eg alginic acid); and glidants (eg magnesium stearate). . Sweeteners and / or flavoring agents are used when administration in liquid form is desired, and can be given intravenously by dissolving in isotonic saline or phosphate buffer.

The peptide should be administered under the guidance of a doctor,
The pharmaceutical composition will then usually contain the peptide together with conventional pharmaceutically or veterinary acceptable carriers.
Generally, the dose will be about 1 to 200 peptides / kg of animal body weight.
It will be in the μg range. In some cases, treatment of patients with these peptides replaces ACTH or corticosteroids, in which case about 10 ng / kg body weight is used.
Moderately low doses are used. All temperatures as used herein are in ° C and ratios are by volume. The percentage of liquid substance is also on a volume basis.

While the present invention has been described in terms of the best mode of implementation currently known to the inventor, it will be apparent to those of ordinary skill in the art that various changes and modifications are covered by the claims appended hereto. It will be understood that it can be done without departing from the scope of the invention described in. For example, substitutions and modifications at other positions in the CRF peptide chain can be made by current or future development without diminishing the potency of the peptide analog. The amino acid sequences from positions 4 to 41 present in the synthetic peptides of the invention appear to be important, while the rest of the peptide molecule does not appear to be particularly limiting. For example,
Instead of a simple amide at its end, a lower alkyl-substituted amide (eg, methylamide, ethylamide, etc.) may be introduced. Similarly, 1-10 additional amino acid residues can be introduced at the N-terminus of the peptide without adversely affecting the biological potency of the peptide.
Such peptides are considered to be included within the scope of this invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Wylie Walker Vale Giunia-California, USA 92037 La Holla Valdez 1643 (56) References Proc. Natl. Acad. Sc i. U. S. A. , 80 (15), 4851-4855 (1983) EMBO J. , 2 (5), 775-779 (1983)

Claims (2)

[Claims] 1. The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
A synthetic peptide represented by Met-Glu-Ile-Ile-NH ₂ or a non-toxic salt thereof. 2. The following formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-
His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Gl
u-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-
Mammalian ACTH and β-endorphin-like substance containing a synthetic peptide represented by Met-Glu-Ile-Ile-NH ₂ or a non-toxic salt thereof, and a pharmaceutically or veterinarily acceptable liquid or solid carrier A pharmaceutical composition for promoting the secretion of erythrocyte.